What is the name of two dyes used in negative staining?

Nigrosin and india Ink.

You are observing Streptococcus pneumoniae on a slide that has been stained using the capsular staining method. However, you do not see capsules surrounding the cells as you expected. What is a likely reason for this?

The slide was heat fixed, which can shrink or destroy capsules.

Capsule staining is different from other classic microbiology staining protocols because?

1. Heat fixing is omitted

2. Capsules are specialized microorganism structures and require different techniques to visualize.
3. Capsule staining is a negative-staining protocol, compared to many other staining protocols that are positive staining.

Which are the primary and secondary dyes for pore staining?

1. Malachite Green (Primary stain)

2. Safranin (Secondary stain)

What are the steps for pore staining?

1. Apply Malachite Green with small piece of paper

2. Heat fix

3. Rinse

4. Apply Safranin

5. Rinse

If safranin was omitted from the spore stain, the vegetative bacterial cells would appear?

Clear

What is the purpose of the heating step?

it allows the dye to penetrate the pores.

What is the primary stain for acid fast staining?

Carbolfuchsin

What is the secondary stain in acid fast staining?

Methylene Blue

What are the steps in acid fast staining?

1. Primary stain 5min

2. Decolorizer 1 min

3. Counterstain 30 min

Rinse with water in between

What type of staining is acid fast staining?

it is a differential stain.

What does Carbolfuchsin bind to?

Mycolic acid in the cell wall

You are given an unknown species of Staphylococcus growing in agar. How would you identify it?

I will use characteristics such as hemolysis, coagulase, novobiocin resistance and mannitol fermentation to determine identity of each of the unknowns.

Which type of Agar will you use to detect for fermentation, hemolysis and novobiocin resistance?

1. Mannitol (MSA)-Fermentation

2. Sheeps' blood Agar (SBA)-Hemolysis

3. In order to determine if your unknown is resistant or sensitive to Novobiocin, you will first have to set up a resistance assay using a Novobiocin disk.

The coagulase test can be performed using two different protocol. What are they?

1.The slide test is relatively easy to do giving results in less than 30 seconds. However, the slide test can lead to false negatives.

2.The test tube protocol is considered the definitive test with easy to read positive and negative results within 24 hours.

What is the expected result of the coagulase test?

For both tests, clumping and solidification of the liquid rabbit plasma indicates a positive result.

The first step in identifying a microorganism is to observe which culture media it will grow on. T/F

True

The next step after identifying media for a microorganism to be identified is to determine what conditions the microorganism grew under such as: aerobic, anaerobic, or aerotolerant. T/F

True

Viewing a Gram stain under the brightfield microscope is a skill that takes practice. To correctly interpret a Gram stain, you should start with the 10X objective to locate and focus on the cells and then work your way to the 100X immersion oil objective to see more details. Record your bacterial morphology observations based on what characterisitics?

•Gram stain reaction

•Cell morphology or shape

•Cell arrangement

The third step to identify a microorganism is to observe colony characteristics. T/F

True

What are the colony characteristics that should be observed?

1. Shape, size, color, surface appearance, and
texture. A

2. Hemolysis.

What are the bacterial colony sizes?

Bacterial colonies can vary in size from:

1. Pinpoint (<0.5mm - however, this size is usually only noted when identifying beta-hemolytic Streptococci)

2. •Small (<1mm)

3. Medium (approx. = 1mm)

4. large (>1mm)

What testing needs to be done for confirmation of S. aureus isolation ?

Coagulase testing

Biochemical tests are comprised of three methods, what are they?

spot, rapid, and conventional procedures.

What is the catalase test detect for?

to detect the presence of catalase enzymes, which hydrolyzes hydrogen peroxide into water and gaseous oxygen (bubbles form)

What does the catalase differentiate for?

This test is useful in differentiating Streptococcus spp. from Staphylococcus spp.

What is the interpretation for a catalase test?

1. Positive reaction = immediate formation of bubbles

2. Weak positive reaction = formation of 1 or 2 bubbles

3. Negative reaction = no formation of bubbles or few bubbles after 20 seconds

What is the bile solubility test based on?

The bile solubility test is based on the observation that pneumococcal cells lyse in the presence of bile salts (sodium desoxycholate).

What is the interpretation for the bile solubility test?

1. Soluble = flattening or disintegration of the colony within 30 minutes

2. Insoluble = no change in colony integrity within 30 minutes

What does the bile test differentiate for?

This test is useful to differentiate Streptococcus pneumoniae (positive) from alpha-hemolytic Streptococcus spp. (negative).

What does UV light do to bacteria?

1. UV light controls bacterial growth by causing irreparable levels of damage to the bacterial cell DNA.
2. UV-light is a form of non-ionizing radiation that creates thymine-thymine dimers which in turn disrupts DNA replication.

What are the steps to the Kirby-Bauer test?

1. Create a “lawn” of bacteria on your MHA plate:

2. Placing the antibiotic disks - 2 options use the antibiotic disk dispenser” or apply each disk by hand

3. Analyzing results.

What is Mueller Hinton agar?

A special agar, Mueller-Hinton agar, is used along with antibiotic
disks containing known standardized concentrations of antibiotic.

How does one make bacterial lawn?

a. Drag the swab over the surface of the agar plate.
b. Rotate the plate 1/3 of a turn and swab surface
again.
c. Repeat 1 or 2 more times until the entire surface
has been swabbed (do not leave any unswabbed
areas).
d. Dispose of the swab in the biohazard container

What effect would endospore formation have on this UV light? Explain fully

UV light cannot penetrate endospores.

What is the coagulase protocol?

1. Draw two circles on the slide using a waxed pencil.
2. Using a sterile pipet, place a drop of sterile water into one of the wax circles. The water is a
control to verify that the strain does not display auto-agglutination (a false positive).
3. Using a sterile pipet, place a small drop of reconstituted rabbit plasma into the other wax
circle (or have your instructor provide you with the plasma sample).
4. Using the stick end of a sterile swab, collect cells from one colony and mix/emulsify the cells
in the water. Repeat in the drop of rabbit plasma. Remember to biohazard swabs after use.
5. Watch for clumping within 15-30 seconds of adding the bacterial cells to the plasma. The
clumping will become more visible if each sample is rocked gently (careful not to spill

What is the bile esculin test?

The bile esculin test identifies if a microorganism can hydrolyze esculin in the presence of bile.

What is the Bile esculin identify?

Bile esculin agar can be used to presumptively identify group D streptococci, enterococci, and Listeria.

What is the interpretation of the bile esculin test?

1. Positive = blackening of the media

2. Negative = no color change of the media

Why shoud the coagulase test be read within 20 minutes?

After that there will be false positives.

What is the tube coagulase test?

The tube coagulase test is a 4 to 24 hour test that detects clumping factor and free coagulase. This test should be performed as confirmation to the slide test.

What is the interpretation of the tube coagulase test?

1. Positive result = plasma will coagulate and form a gel (sometimes the plasma will solidify)

2. Negative result = plasma will remain a liquid

How are streptococcus sp catergorised in a catalase test?

Streptococcus spp. are categorized by the type of hemolysis they produce on BAP (Alpha, Beta, and Gamma).

What is the next step after identifying S.aureus?

determine if the microorganism is methicillin-resistant and if it is the same strain as the patient isolates. This would require susceptibility testing to check for methicillin resistance and PFGE testing for DNA typing and comparison.

What is the Interpretation of CHROMagar MRSA test?

MRSA colonies on CHROMagar MRSA will appear mauve. Other microorganisms (non-MRSA) will be inhibited or produce colorless, white, blue or blue/green colonies.

You have a Gram positive microorganism. Which biochemical test is useful in the differentiation of Staphylococcus spp. from Streptococcus spp.?

Catalase test

Streptococcus spp., such as Streptococcus pyogenes (Group A Strep) and Streptococcus pneumoniae, are the most common aerobic, catalase negative, Gram positive cocci that can cause disease in humans. It is critical that you identify these microorganisms quickly as they may cause life threatening illnesses such as pneumonia, meningitis, sepsis, and necrotizing fasciitis. T/F

True

Streptococcus spp. are Gram positive cocci found in pairs or chains. On blood agar (BAP), Streptococcus spp. usually produce glistening, smooth colonies.

True

Why is it important that you are able to differentiate the beta-hemolytic streptococci?

1. Streptococcus pyogenes (Group A Strep) causes: Strep throat, pneumonia, impetigo, tissue infections, pleuritis, sepsis, and necrotizing fasciitis.

2. Streptococcus agalactiae (Group B Strep) is important as it is a leading cause of meningitis in newborns. Because of this, clinical laboratories screen pregnant women to see if they are carriers.

CoNS are recognized as a cause of nosocomial infections in hospitals. T/F

True

In a Gram stain, Staphylococcus spp. are Gram positive cocci found in clusters. On BAP, Staphylococcus spp. usually produce large, white-yellow or grey creamy, smooth colonies. S. aureus is beta-hemolytic while CoNS are usually non-hemolytic. T/F

True

Lactobacillus are; medium, straight, uniform Gram positive rods, rounded ends that may be coccobacillary may form chains or spirals. T/F

True

Corynebacterium are:Gram positive, pleomorphic, club shaped, rods, or coccobacilli that form palisading and/or angular arrangements. T/F

True

Isolation of Streptococcus is typically done using blood agar. Note: SBA is differential for RBC hemolysis but is not selective for
Streptococcus species. T/F

True